tbuckley/cabot-search huggingface dataset:
P19 Inducible 14-3-3 sigma/stratifin ablation accelerates malignant progression in HK1.ras/fos-Δ5PTENflx transgenic mouse skin carcinogenesis
Authors: Imogen Rawlinson, Carol McMenemy, David Greenhalgh
Journal: British Journal of Dermatology (2023)

Abstract In human carcinogenesis, given the plethora of pathways associated with 14-3-3σ and their complex interactions, the causality of 14-3-3σ deregulation remains elusive, with both tumour suppressive and oncogenic roles. To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of endogenous 14-3-3σ (stratifin) was analysed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of phosphatase and tensin homolog (PTEN)-mediated Akt regulation (K14.creP/Δ5PTENflx) and via conditional RU486-inducible 14-3-3σ knockout (K14.creP-Δ14-3-3). Consistent with 14-3-3σ roles in the commitment of keratinocytes to differentiate, bigenic HK1.fos/Δ5PTENflx hyperplasia expressed 14-3-3σ in basal layers and paralleled keratin K1 expression which appeared alongside elevated p53/p21 to increase keratinocyte differentiation leading to a keratoacanthoma (KA) aetiology. Trigenic HK1.ras/fos-Δ5PTENflx hyperplasia/papillomas also displayed increased basal-layer 14-3-3σ, suggesting attempts to protect basal layer p53 tumour suppressor gene function, given that 14-3-3σ acts as a chaperone protein to remove/relocate MDM2 for degradation, thus maintaining p53 levels. With time, HK1.ras/fos-Δ5PTENflx papillomas exhibited reduced p53 and increased p-MDM2166 activity in basal layers, which coincided with malignant conversion. Surprisingly, despite this p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCC) and, alongside elevated p21, appeared to limit further malignant progression via inhibiting p-Akt1473 expression. To further assess causality, 14-3-3σ functions were ablated in this model via an RU486-inducible cre/loxP system expressed from a keratin 14 (K14) promoter (K14.creP-Δ14-3-3σ). In bigenic HK1.fos-Δ14-3-3σ, preliminary data suggest loss of 14-3-3σ functions resulted in increased hyperplasia and keratosis similar to that observed in bigenic HK1.fos/Δ5PTENflx hyperplasia. Functional 14-3-3σ ablation in HK1.ras.fos/Δ5PTENflx/Δ14-3-3σflx genotypes apparently failed to accelerate papillomatogenesis, suggesting elements of redundancy in terms of tumour promotion; however, loss of 14-3-3σ facilitated increased p-MDM2166 and p53 loss, resulting in malignant conversion with progression to aggressive SCC. Collectively, these data suggest that 14-3-3σ/stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms, while persistent expression in wdSCCs suggests p53-independent scenarios that may involve p21-mediated Akt1 inhibition. However, if ablated, this limit of early-stage malignant progression is lost and 14-3-3σ loss leads to rapid progression to aggressive, p21-negative SCCs exhibiting uniform p-Akt1473activation.
O09 Dynamic impact of chronic inflammation on epidermal autophagy pathway
Authors: Nazia Uddin, Paola Ostano, Giovanna Chiorino, Jeremy di Domizio, Michel Gilliet, Mike Philpott, Rosalind Hannen, Daniele Bergamaschi
Journal: British Journal of Dermatology (2023)

Abstract Chronic inflammatory signalling significantly affects epidermal homeostasis leading to different types of skin conditions characterized by a deregulated stratification of epidermal keratinocytes. Our project aims to investigate how chronic inflammation mechanistically influences autophagy signalling in the epidermis. We have previously reported impairment of the autophagic flux in an established psoriatic keratinocyte model (PSA cells) when compared to corresponding controls from healthy skin (KT2 and NTERT cells). To understand the dynamic impact of chronic inflammation on the epidermal autophagy pathway we have custom made a cytokine cocktail which induces interleukin-1β release at different times in keratinocyte cultures and stimulates keratinocyte proliferation. Stimulation of healthy keratinocytes with chronic inflammatory cocktail-induced activation of initial steps of the autophagy process. This is confirmed by the protein expression of key components of ULK1 and VPS34 complexes. Analysis of the elongation-stage markers confirmed the activation and correct proceeding of the pathway, until we observed LC3-I accumulation. In autophagy, the phospholipid phosphatidylethanolamine (PE) is crucial for LC3-I conversion to LC3-II. Changes in phospholipid metabolism have been reported in inflammatory skin conditions, including PE downregulation in psoriatic keratinocytes. By increasing PE expression in inflamed keratinocytes, we show increased LC3-II lipidation and restoration of the autophagy pathway functionality. Moreover, inflammation-induced p62 upregulation (a cargo degraded by the lysosomal autophagy compartment) and deregulated the lysosomal membrane permeability integrity, as confirmed by the expression of glycosylated and deglycosylated lysosomal protein (LAMP1, LAMP2 and LAMP3) expression. Our data are supported by independent transcriptomic data analysis from the GEO database, comparing autophagy gene expression in psoriatic and healthy matched or nonmatched controls. Spatial transcriptomic mapping of autophagic genes in lesional psoriatic skin provided further insight into the epidermal autophagy pathway regulation in inflammatory skin conditions. Immunofluorescence staining of both healthy and psoriatic human skin samples finally translate the differences in key autophagy marker expression observed in our two- and three-dimensional cell models, including deregulation of LC3, p62 and lysosomal membrane protein expression. Overall, our results exhibit a comprehensive and mechanistical analysis of how chronic inflammation could affect the integrity of this metabolic pathway, which significantly regulates epidermal homeostasis. Our data provide novel targets for therapeutic strategies and supports autophagy stimulation as viable modes of treatment to counteract chronic inflammatory skin conditions.
P27 Uncovering the roles of lysosomal protease cathepsin H in atopic dermatitis
Authors: Theresa Tachie-Menson, Atqiya Azizah, Aaron  Hughes, Ryan O'Shaughnessy
Journal: British Journal of Dermatology (2023)

Abstract Atopic dermatitis (AD) is a persisting skin condition characterized by recurring eczematous lesions and pruritus. It is ranked as a nonfatal disease with the fifteenth-highest global burden. The causes of AD are diverse: a defective epidermal barrier, epidermal inflammation, an altered epidermal microbiome and genetics. However, much of the treatment for AD focuses on reducing inflammation. Further understanding on how the epidermal barrier maintains its integrity may be useful in developing treatments that specifically help to restore the defective epidermal barrier. We have identified cathepsin H, a lysosomal protease with aminopeptidase activity, as a protease with key roles in maintaining the integrity of the epidermal barrier. Ctsh+/– mice exhibit reduced corneocyte integrity, a reduction in the expression of the cornified envelope protein loricrin, reduced filaggrin processing and altered skin immunity, among other defects. These highlight the significant roles of cathepsin H within the epidermal barrier and terminal differentiation process of epidermal keratinocytes. To investigate the roles of cathepsin H in the epidermal barrier we have used patient biopsies, patient tape-strip samples and rat epidermal keratinocytes (REKs), a keratinocyte cell line that spontaneously undergoes differentiation and recapitulates the complete differentiation process of human epidermal keratinocyte. Using REKs, we have generated Ctsh knockdown cell lines, from which we have derived three-dimensional organotypic cultures, as well as cathepsin H and cathepsin B stable overexpression cell lines. Moreover, we have performed mass spectrometry analysis of tape-strip samples from patients with AD exhibiting a high or low expression of cathepsin H. Our research aims to uncover the protein targets of cathepsin H in the epidermis and its ensuing functions in the epidermal barrier.
P30 Microsatellite instability in cutaneous squamous cell carcinoma
Authors: Georgie Holt, Christine Hayes, Rachel  Phelps, Akhtar Husain, Christopher Lamb, Richard  Gallon, Neil Rajan
Journal: British Journal of Dermatology (2023)

Abstract Lynch syndrome (LS) is a hereditary cancer syndrome caused by pathogenic germline variants in genes that encode mismatch repair (MMR) proteins. LS is characterized by an increased risk of multiple cancers, including colorectal and endometrial cancer. Muir–Torre syndrome (MTS) is an allelic, phenotypic variant of LS characterized by the presence of skin tumours, including keratoacanthomas and sebaceous tumours. Recently, an association has been proposed between MTS/LS and cutaneous squamous cell carcinoma (cSCC). MTS/LS tumours demonstrate a mutation signature termed microsatellite instability (MSI) that can be used to screen sporadic tumours for underlying MTS/LS. We have previously shown that amplicon-sequencing of microsatellites can detect increased MSI in cSCC in patients with LS. In this study, we assayed a pilot cohort sporadic cSCC in the general population to further explore this potential association. DNA was extracted from cSCC samples. We used an established molecular inversion probe-based protocol to amplify informative microsatellites and sequenced these using the Illumina MiSeq platform. An MSI score was calculated for each tumour sample using a naïve Bayesian approach based on microsatellite deletion frequencies. We found that 5% (n = 1/19) of cSCCs were MSI-high (95% confidence interval 0.1–26.0), which is equivalent to estimated frequencies among unselected sporadic cSCCs in recent literature. We plan to expand our pilot cohort to include a larger series of cSCC and to also carry out somatic sequencing of MSH2, MSH6, MLH1 and PMS2 in tumours to determine if MSI status correlates with MMR gene variant status. Ultimately, this work aims to improve identification of new patients with LS presenting with cSCC and allow them to benefit equitably from preventative cancer surveillance and aspirin chemoprevention.
O22 Programmed cell death-1 receptor is a checkpoint in Tbet+ Th17 cells within the tumour microenvironment
Authors: Katie Smith, Jing Xuan Lim, Arian Laurence, Shoba Amarnath
Journal: British Journal of Dermatology (2023)

Abstract Programmed cell death-1 (PD-1) receptor is critical for immune regulation and is expressed on a variety of immune cells, including effector T cells. In tumours, PD-1 engagement dampens immune activation and increases tumour progression. However, it is unclear how PD-1 affects T helper (Th) cell differentiation, namely Th17 cells. Previously, we have demonstrated that PD-1 maintains T regulatory (Treg) cell plasticity, and Tbet+ Th1 cell plasticity to Treg cells in inflammation and cancer. Therefore, it is clear that PD-1 is indispensable in T-cell lineage maintenance, with limited understanding on its role in Th17 differentiation, function and lineage stability. We hypothesized that as PD-1 blockade augments Th17 responses in cancer, PD-1 could control Th17 function and lineage stability within the tumour microenvironment. We established a B16 F10 melanoma model in wild-type (WT) and Pdcd1–/– (PD-1) mice, and isolated Thy1+ tumour-infiltrating lymphocytes (TILs) from tumours. Single-cell analysis was conducted, and protein and gene expression analysed within the RORc cluster. Our results demonstrated that WT and PD1–/– mice possessed RORc-expressing cells within the TILs, with PD1–/– mice showing an increased number of Th17+ TILs, revealing a role for PD-1 in regulating Th17 responses within TILs. We next investigated the RORc cluster for function and found expression of Il17a and Ifng. Of note, we found that Th17 differentiation genes [Dusp2 (P = 0.004); Ddx58 (P = 0.009)] were significantly regulated by PD-1. Although an increase in interleukin (IL)-12RB1 (P = 0.001) and IL-17RB protein (P = 0.004) was noted, suggesting a plasticity towards either Th1 or Th2 phenotypes, a significant downregulation of Tbet (Tbx21, P = 0.003) was observed in the PD1–/– population. These data suggest that PD-1 is required for RORc+ cells to acquire functional plasticity within tumours, thereby highlighting a role for PD-1 in Th17 plasticity. In summary, our study is the first to show the importance of PD-1 in driving Tbet+ Th17 cell differentiation within the TME.
O07 Filaggrin insufficiency renders keratinocyte-derived small extracellular vesicles capable of affecting CD1a-mediated T-cell responses and promoting allergic inflammation
Authors: Adrian Kobiela, Weronika Hewelt-Belka, Joanna E Frąckowiak, Natalia Kordulewska, Lilit Hovhannisyan, Aleksandra Bogucka, Rachel  Etherington, Artur Piróg, Irena  Dapic, Susanne Gabrielsson, Sara Brown, Graham S   Ogg, Danuta Gutowska-Owsiak
Journal: British Journal of Dermatology (2023)

Abstract The association between FLG loss-of-function mutations and the development of atopic dermatitis (AD) indicates that filaggrin is critical to skin barrier function; the mutations are also linked to additional allergic manifestations, but it is unknown how the skin may influence inflammation in distant tissues. Here we investigated the impact of filaggrin insufficiency on keratinocyte-derived exosome-enriched small extracellular vesicles (sEVs) and found that those secreted by filaggrin-knockdown cells are extensively remodelled as a consequence of the abnormal keratinocyte differentiation process. The altered sEV lipidome changes the sEV capacity to promote T-cell responses by direct effect on self-lipid neoantigen generation, modulating the content of permissive (stimulatory) and nonpermissive (inhibitory) CD1a ligands released from the sEV membranes by phospholipase A2. Presentation of those lipids to T cells results in the redirection from a type 1 to a type 2 response. We found that the aberrant sEV lipid composition mirrors a generalized remodelling of cellular lipids resulting from dysregulated expression of multiple enzymes of lipid metabolic pathways, observed both in filaggrin knockdown keratinocytes in vitro and in the skin of patients with AD. Hence, filaggrin insufficiency background renders keratinocytes capable of supplying CD1a ligands which are capable of impeding both homeostasis-promoting and protective antimicrobial responses, enhancing type 2 T-cell activation to perpetuate inflammation in AD skin. sEV transfer by systemic circulation may promote similar effects in distant tissues.
O19 CYLD cutaneous syndrome tumours demonstrate increased NF-κB signalling and diminished collagen organisation.
Authors: Joseph Inns, Neil Rajan
Journal: British Journal of Dermatology (2023)

Abstract CYLD is a tumour-suppressor gene encoding a deubiquitinase, targeting K63- and M1-linked ubiquitin chains and thereby influencing a variety of cellular processes. Loss of function in patients with CYLD cutaneous syndrome (CCS) results in the formation of benign cutaneous cylindromas, spiradenomas and trichoepitheliomas. These CCS skin tumours originate from the hair follicle and appear from adolescence onwards. We used an unbiased proteomics approach to analyse CCS tumour and control samples (n = 8). Label-free quantification of 627 proteins revealed significant changes in 139 proteins with a fold change &amp;gt; 1.75 and P-value &amp;lt;0.05, between control and tumour samples. Gene Ontology analysis identified upregulation of nuclear factor-κB (NF-κB) and immune signalling proteins alongside an increased prevalence of spliceosome proteins in tumour samples. Collagen 7, collagen 4, agrin and thrombospondin-1 were increased in tumour samples, providing, for the first time, an unbiased set of extracellular matrix (ECM) proteins generated by CCS tumour cells. Conversely, CCS tumour samples demonstrated a decreased abundance of proteins associated with collagen organization and fibril formation compared to normal skin. Further analysis of CCS tumours through immunoblotting revealed a reduction of collagen I, while collagen VII was highly upregulated in both tumour lysate and secretome. Our findings indicate that loss of CYLD function impacts on cellular signalling pathways such as NF-κB signalling, and is associated with hypersecretion of select ECM proteins.
P06 Functional MDM2 ablation in HK1.ras/fos-Δ5PTEN transgenic mice results in an elevated, p53-mediated block of carcinogenesis that depends upon p53 expression
Authors: Sofia Moula, Gavin Turnbull, David  Greenhalgh
Journal: British Journal of Dermatology (2023)

Abstract MDM2 is a ubiquitin ligase that targets p53 for degradation and the efficacy of functional MDM2 knockout to protect p53 levels was shown to inhibit benign papilloma formation in HK1.ras transgenic mice. In skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of phosphatase and tensin homolog (PTEN)-mediated Akt regulation, p53 loss was a consistent theme leading to well-differentiated squamous cell carcinoma (SCC). Analysis of endogenous MDM2 status in HK1.ras/fos-Δ5PTENflx hyperplasia and papillomas showed that activated p-MDM2166 activity was initially confined to suprabasal layers with resultant elevated basal layer p53. However, with time, HK1.ras/fos-Δ5PTENflx papillomas exhibited reduced p53 levels in basal keratinocytes associated with increased p-MDM2166 activity, which coincided with malignant conversion, suggesting that suprabasal-to-basal increases in p-MDM2166 downregulated p53 giving a susceptibility to malignant conversion. Therefore, to assess whether removal of this MDM2 ability to degrade p53 could inhibit carcinogenesis in HK1.ras/fos-Δ5PTENflx mice, MDM2 exons 5 and 6, which bind p53 and chaperone for ubiquitination, were removed via a Cre/lox-P system driven by a keratin K14 promoter following treatment with topical RU486 (K14cre-Δ5/6MDM2flx/flx). RU486 treatment of HK1.ras/fos-Δ5PTENflx-Δ5/6MDM2flx/flx mouse skin resulted in a complete block of carcinogenesis in all animals for over 16 weeks due to elevated p53 expression. However, preneoplastic hyperplasia appeared to be relatively unaffected, despite excess p53 expression. In approximately 10–15% of animals, HK1.ras/fos-Δ5PTENflx-Δ5/6MDM2flx/flx mice developed overt tumours by 5–6 months. A similar finding was observed in HK1.fos-Δ5PTENflx keratoacanthoma (KA) aetiology, as here the early p53 expression in HK1.fos-Δ5PTENflx-Δ5/6MDM2flx/flx hyperplasia mediated by MDM2 loss also blocked KA aetiology. To assess whether this was specific to MDM2/p53 interactions, inducible p53 ablation was introduced into the HK1.fos-Δ5PTENflx KA model. This confirmed inhibition was specific to p53 expression, as all HK1.fos-Δ5PTENflx/p53 flx/flx tumours rapidly progressed to malignancy, regardless of MDM2 genetic status. Thus, MDM2 inhibition can limit carcinogenesis via maintaining p53 levels; however, this inhibition can be circumvented via p53 loss and, moreover, MDM2 inhibition would be counterproductive in SSC contexts that expressed gain-of-function p53 mutations.
O04 HPV8 E6 leads to Lrig1+ keratinocyte stem cell expansion
Authors: Boris Shorning, Huw J Morgan, Carlotta Olivero, Cinzia Borgogna, Marisa Gariglio, Baki Akgul, Girish K Patel
Journal: British Journal of Dermatology (2023)

Abstract Actinic keratoses (AKs) represent the precursor skin lesions in the pathogenesis of up to 82% of cutaneous squamous cell carcinoma (cSCCs). However, current treatment of AK lesions leads, at best, to only a 75% reduction in cSCCs for 1 year, and relies on therapies using topical chemotherapy, immune stimulants or surgical destruction. We have previously shown that 44% of AK tissue sample histologies contain koilocytes, and genotyping universally identified human papillomavirus 8 (HPV8). To determine the cell signalling basis for HPV8 Lrig1 hair follicle junctional zone keratinocyte stem cell proliferation and expansion, we sought to identify the integral early region gene(s). Skin histology of HPVtg mice expressing individual early-region genes (E2, E4, E6 and E7) identified both E2 and E6 mouse skin as having broadened hair follicle infundibulum. Whole-mount immunofluorescent labelling demonstrated Lrig1 proliferation and expansion in E6 mice only, which we confirmed by flow cytometric analysis. Compared to wild-type (WT) mice, E6 mice Lrig1 flow-sorted cells had lower expression of the differentiation marker keratin 10 but comparable levels of Sox9 and cMyc, consistent with expansion of keratinocyte stem cells, as confirmed by colony-forming efficiency assay (WT 0.05 ± 0.043 vs. E6 0.24 ± 0.11). E6-transduced, but not E7-transduced, HaCaT keratinocytes demonstrated reduced differentiation and increased p63 expression with induced differentiation using calcium shift. Compared to vector control and E7-transduced, E6-transduced HaCaT keratinocytes demonstrated increased migration and colony-forming efficiency but similar proliferation, consistent with greater expression of pSTAT3 and ΔNP63. Ingenuity pathway analysis of previously determined E6 binding partners identified p300 as a regulator of STAT3 activation. Small interfering RNA targeting P300 in E6-transduced HaCaT cells led to a reduction in pSTAT3 and ΔNP63. Pull-down experiments show that E6 bound acetylated p300, which, in turn, led to increased expression of STAT3 regulated genes. The E6K137N mutation in transgenic mice, wherein E6 does not bind p300, do not develop papilloma or cSCC. Our findings show that E6, not E7, as often observed in α-serotype HPV, was responsible for HPV8-induced Lrig1 hair follicle junctional zone keratinocyte stem cell expansion.
P33 Investigating the utility and exploring biological targets of micro-wound healing compounds in 2D and 3D skin models
Authors: Metka Gorkic, David Messenger, Linda  Wainwright, John Connelly, Cleo L   Bishop
Journal: British Journal of Dermatology (2023)

Abstract An injury to the epidermis can result in a micro-wound, characterized by its localization above the dermal–epidermal junction and an absence of vessel damage. Re-epithelialization is driven by keratinocytes, which organize into overlapping migrating and proliferating zones to ensure faithful micro-wound closure. Although still relatively unexplored, keratinocyte-mediated cytokine secretion is thought to be a feature of the micro-wound re-epithelialization response. A failure to re-epithelize, also characteristic of chronic wounds, can lead to reoccurring infections and hyperpigmentation. Although wound healing has been extensively researched, there are gaps in our understanding of the cellular mechanisms involved in the onset and completion of re-epithelization, leading to a scarcity of available enhanced micro-wound healing compounds (MHCs). To investigate the utility of a panel of putative MHCs, a new high-throughput scratch assay method was devised. A guide for multichannel pipette scratches was produced by three-dimensional printing an autoclavable 96-well plate lid with custom-made lengthwise slits. Using an immortalized keratinocyte cell line (N/TERT), the rate of scratch healing was characterized. Critical re-epithelization time points have been determined, and markers of migration are being explored within the identified periods. Moreover, known keratinocyte migration-inducing compounds are being tested to serve as a positive control for enhanced MHC screening. Any putative MHC hits will be further explored using proteomic approaches to elucidate the signalling pathways involved in the onset and completion of re-epithelization. This analysis will assist in the identification of novel enhanced MHC targets and further our understanding of keratinocyte re-epithelization.